Bin1 expression as a marker of cancer

ABSTRACT

Provided are methods for screening a subject for cancer. The methods involve obtaining a blood sample from the subject and determining a level of Bridging Integrator 1 (BIND isoforms comprising exon 12a in the sample. Optionally, the method involves determining a level of 12a+/13− BIN isoform (comprising exon 12a but lacking exon 13) in the sample. An elevated level of 12a+ (e.g., 12a+/13−) BIN1 isoforms in the blood sample indicates the subject has cancer. Also provided are methods for determining efficacy of a cancer therapy in a subject and methods of treating cancer. Isolated antibodies that selectively bind human 12a+ BIN1 are also provided as well as kits for determining 12a+/13− BIN1 isoforms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.61/541,539, filed Sep. 30, 2011, which is hereby incorporated herein byreference in its entirety.

BACKGROUND

Cancer is one of the leading causes of death in the United States. Earlydiagnosis of cancer and effective monitoring of metastasis and treatmenteffects can assist in risk stratification and in guiding therapy.

SUMMARY

Provided are methods of screening a subject for cancer. The methodscomprise obtaining a blood sample from the subject and detecting in thesample a level of Bridging Integrator 1 (BIN1) isoforms that containpolypeptide encoded by exon 12a (i.e., 12a+ BIN1). An elevated level of12a+ BIN1 isoforms in the blood sample, as compared to a negativecontrol level, indicates the subject has cancer. Therefore, if thesubject has an elevated level of 12a+ BIN1 isoforms, the method canfurther comprise obtaining a tissue sample from the subject, e.g., forhistological examination or other analysis for the purpose of confirmingand further defining the cancer.

There are at least five isoforms of BIN1 that contain polypeptidesencoded by exon 12a: isoforms 1, 4, 5, 6 (also referred to herein asCa-1), and the isoform referred to herein as Ca-2. The disclosed methodcan therefore involve determining the level of a subset of BIN1isoforms, including the levels of Ca-1, Ca-2, or a combination thereof.Therefore, the disclosed method can involve determining the Ca-1 and/orCa-2 isoform level in the blood sample.

Also provided are methods for determining efficacy of a cancer therapyin a subject based on changes in levels of the Ca-1 and Ca-2 BIN1isoforms. The methods can therefore comprise obtaining a first bloodsample from a subject with cancer prior to treatment with a first cancertherapy, determining a first level of Ca-1 and/or Ca-2 isoform in thefirst blood sample, obtaining a second blood sample from a subject withcancer after at least one treatment with the first cancer therapy,determining a second level of Ca-1 and/or Ca-2 isoform in the secondblood sample, and comparing the first level to the second level. Inthese methods, if the Ca-1 and/or Ca-2 isoform value increases or failsto decrease in the second blood sample as compared to the first bloodsample, a second cancer therapy can be selected for the subject, whichincludes supplementing or replacing the cancer therapy with additionalor alternative surgery, chemotherapy, or radiation therapy. For example,dosage of a chemotherapeutic can be increased. If the Ca-1 and/or Ca-2isoform value decreases in the second blood sample as compared to thefirst blood sample, treatment of the subject with the first cancertherapy can be continued. If the Ca-1 and/or Ca-2 isoform value issufficiently reduced, therapy may be discontinued or maintenance therapyinitiated. This method can be repeated for each subsequent cancertherapy. The level of the Ca-1 and/or Ca-2 isoforms is an indication ofcancer burden, thereby allowing for quantification of disease andeffectiveness of cancer therapy.

Further provided are methods of treating cancer in a subject. Themethods comprise determining levels of Ca-1 and/or Ca-2 isoform in afirst blood sample from a subject with cancer, providing a firsttreatment to the subject, determining levels of Ca-1 and/or Ca-2 isoformin a second blood sample from the subject, and providing a secondtreatment to the subject based on whether the level of Ca-1 and/or Ca-2isoform in the second blood sample is higher, lower, or the same as thelevel of expression in the first blood sample.

Also provided is a method of treating cancer in a subject by selecting asubtype of cancer showing an elevated Ca-1 and/or Ca-2 isoform level andproviding a therapy that addresses the BIN1 pathway. The methodscomprise obtaining a blood sample from the subject, determining a levelof Ca-1 and/or Ca-2 isoform in the blood sample, comparing the Ca-1and/or Ca-2 isoform level to one or more control levels, andadministering to the subject an inhibitor of indoleamine 2,3-dioxygenase(IDO) if an elevated Ca-1 and/or Ca-2 isoform level is determined.

Also provided is an isolated antibody that selectively binds thepolypeptide encoded by exon 12a of human BIN1 (12a+ BIN1). Kitscontaining this antibody are also provided for detecting Ca-1 and/orCa-2 isoform levels. The kit can contain an assay system for detecting12a+ BIN1, an assay system for detecting 12a+/13+ BIN1, and/or an assaysystem for detecting 10+/12a BIN1.

The details of one or more embodiments are set forth in the accompanyingdrawings and the description below. Other features, objects, andadvantages will be apparent from the description and drawings, and fromthe claims.

DESCRIPTION OF DRAWINGS

FIG. 1 shows a histogram showing that serum BIN1 is increased in canineswith advanced stages of cancer. Detection is based on total BIN1. ***P<0.001.

FIG. 2 is a sequence alignment of BIN1 isoform 1 and isoform 4, whichboth contain exon 12a.

FIG. 3 is an illustration of the BIN1 isoforms based on the presence orabsence of exons due to alternative splicing.

FIGS. 4A and 4B are standard curves of BIN1 recombinant protein using anassay to detect 12a+/13+ BIN1 levels (FIG. 4A) and an assay to detect12a+ BIN1 (FIG. 4B).

FIG. 5 is a plot showing 12a+/13+ BIN1 levels (x-axis) and 12a+ BIN1levels (y-axis) in normal (square) and cancer (triangle) samples.

FIG. 6 is a plot showing 12a+/13− BIN1 levels in normal samples as afunction of age.

FIG. 7 is a plot showing 12a+/13− BIN1 levels in human normal, lungcancer, pancreatic cancer, colorectal cancer, ovarian cancer, andthyroid cancer samples. Horizontal bars represent the median.

FIG. 8 is a plot showing 12a+/13− BIN1 levels from combined cancersamples as a function of cancer stage.

FIGS. 9A-9E are plots of 12a+/13− BIN1 levels from lung cancer (FIG.9A), pancreatic cancer (FIG. 9B), colorectal (FIG. 9C), ovarian (FIG.9D), and thyroid cancer (FIG. 9E) samples as a function of cancer stage.

FIG. 10 is a plot showing 12a+/13− BIN1 levels in blood samples ofnormal and cancer dogs (pre- and post-treatments).

FIGS. 11A-11C are graphs of a time course showing 12a+/13− BIN1 levelsin dogs. FIG. 11A shows results for animals #1 to #4 having apre-treatment signal >15. FIG. 11B shows results for animals #5 to #8having a pre-treatment signal <15 but >2. FIG. 11C shows results foranimals #9 to #11 having a pre-treatment signal >2.

DETAILED DESCRIPTION

Methods described herein are based on the finding that cancer can bedetected in a subject by detecting in a blood sample from the subjectelevated levels of a subset of BIN1 isoforms that contain thepolypeptide encoded by exon 12a (12a+ BIN1). An elevated level of 12a+BIN1 isoforms in a blood sample from the subject, as compared to anegative control level, indicates the subject has cancer. Accordingly,12a+ BIN1 expression can be used as a marker to determine a diagnosis ofcancer in the subject, determine the level of progression or metastaticpotential of the cancer in the subject, and to follow the disease in thesubject. Furthermore, 12a+ BIN1 expression can be used to determine thesubtype of cancer in a subject as a means of selecting an effectivetherapy, including for example, an agent that affects the BIN1 pathway.

The Bridging integrator 1 (BIN1) gene encodes several isoforms of anucleocytoplasmic protein through alternative splicing. Ten BIN1isoforms have been identified to date with two isoforms beingubiquitously expressed while others are present only in specifictissues. Among different functions, BIN1 acts as a tumor suppressorthrough binding the oncogenic protein c-Myc. Accordingly, severalstudies have shown a decrease in BIN1 expression during cancerprogression. Interestingly, there is increasing evidence that aberrantsplicing of BIN1 and a consequently increase in the expression ofspecific isoform(s) correlates with cancer progression. Using publicdatabases listed in Table 1, a study was performed to capture thesequence of the isoform 4 of BIN1 to identify germline and somaticmutations that can occur in the BIN1 sequence, and identifyingcorrelations between alternate BIN1 splicing and expression during humandisease progression.

TABLE 1 Databases used for BIN1 analysis ONIM (Online Mendelian OnlineCatalog of Human Genes and Genetic Disorders Inheritance in Man) HGMD(The Human Gene Resource providing comprehensive data on human inheritedMutation Database) disease mutations to genetics and genomic research.GWAS (Genome-wide Used to identify common genetic factors that influencehealth association studies) and disease. TCGA (The Cancer GenomePlatform to search, download, and analyze data sets Atlas) COSMIC(Catalogue Of Store and display somatic mutation information and relatedSomatic Mutations In Cancer) details and contains information relatingto human cancers HPRD (Human Protein Platform to visually depict andintegrate information pertaining Reference Database) to domainarchitecture, post-translational modifications, interaction networks anddisease association for each protein in the human proteome. AlamutApplication that integrates genetic information from different sourcesin one, consistent and convenient environment to describe variants usingHGVS nomenclature and help interpret their pathogenic status. LOVD(Leiden Open Provide a flexible, freely available tool for Gene-centeredVariation Database) collection and display of DNA variations. CancerGEMKB (Cancer An integrated, searchable knowledge base of cancer humanGenomic Evidence-based genome epidemiology and genomic applications incancer care Medicine Knowledge Base) and prevention DGV (Database ofGenomic A curated catalogue of structural variation in the human genomeVariants) GEO (Gene Expression Public functional genomics datarepository supporting Omnibus) Minimum Information About a MicroarrayExperiment- compliant data submissions. KEGG (Kyoto EncyclopediaBioinformatics resource for linking genomes to life and the of Genes andGenomes) environment. Others: NCBI, Ensembl, UnitProtKB, and GeneCards

The BIN1 gene is located on chromosome 2 (2q14) between 127,805,599 and127,864,903 bps (source: NCBI), and comprises 20 exons which can bealternatively spliced to form at least ten different isoforms. The BIN1protein contains distinct domains such as a BAR domain(BIN1-amphiphysin-Rvs167), a phosphoinositide-binding domain, aclathrin-associated protein-binding domain (CLAP), a Myc-binding domain(MBD), and a Src homology 3 domain (SH3) (Prendergast G C, et al.,Biochim Biophys Acta. 2009 1795(1):25-36). The exon 12a encodes a partof the CLAP domain. Four isoforms of BIN1 contain the exon 12a includingthe longest isoform of BIN1 (variant 1; GenBank accession numberAF004015) and BIN1+12a, also named transcript variant 4 (GenBankaccession number AF068918, NM_(—)139346, NP_(—)647596). BIN1+12a lacksfour in-frame exons and has an additional in-frame exon (exon 10) in thecoding region, compared to BIN1 variant 1. A sequence alignment of BIN1variant 1 and variant 4 is provided in FIG. 2. BIN1 variant 1 andvariant are expressed predominantly in the central nervous system.

Several genetic mutations in the BIN1 gene have been associated with themuscle weakness disorder centronuclear myopathy. These mutations includea homozygous 105G-T transversion in the BIN1 gene, resulting in alys35-to-asn (K35N) substitution, and a homozygous 451G-A transitionresulting in an asp151-to-asn (D151N) substitution. In addition, amutation which generates a prematurely terminated BIN1 protein was alsoidentified with a homozygous 1723A-T transversion in the BIN1 gene,resulting in a lys575-to-ter (K575X) substitution. Finally, a homozygous461G-A transition in exon 6 of the BIN1 gene, resulting in anarg154-to-gln (R154Q) substitution was also identified in a patient withautosomal recessive centronuclear myopathy. The isoform 8 of BIN1(GenBank accession number AF068918), a variant which lacks five in-frameexons including exon 12a and has an additional in-frame exon (exon 10)in the coding region compared to BIN1 variant 1, is specificallyexpressed in skeletal muscle. Alternative splicing of this isoform 8,leading to the exclusion of exon 10 (phosphoinositide-binding domain) isassociated with muscle weakness in Myotonic dystrophy. Finally, severalsingle nucleotide polymorphisms (SNPs) have been described in the BIN1gene, including two in exon 12a. No phenotypes have been identified withthese two SNPs.

BIN1 acts as a tumor suppressor through binding to c-Myc andsubsequently repressing its transcriptional activity. Accordingly,attenuated expression of BIN1 is observed in many cases of breast,prostate, lung, brain, and colon cancers. Interestingly, cancer-specificvariants of the ubiquitous isoforms 9 and 10 present an aberrantinclusion of the CNS-specific exon 12a. 12a+ BIN1 isoforms are observedin many tumor cells and tumor cell lines, and represent a commonmissplicing events occuring in human cancer (Prendergast G C, et al.,Biochim Biophys Acta. 2009 1795(1):25-36). For example, these 12a+ BIN1isoforms are aberrantly expressed in melanoma and this alternativesplicing abolishes the tumor suppressor activity of BIN1 allowing c-Mycoverexpression without induction of programmed cell death (Ge K, et al.,Proc. Natl. Acad. Sci. U.S.A. 1999 96(17):9689-94; Xu Q, et al., NucleicAcids Res. 2003 31(19):5635-43).

Somatic mutations in the BIN1 gene, both missense and synonymous, havealso been reported in several cases of cancer including skin (in 3 outof 8 samples), brain (2/469), lung (1/11), ovary (3/3), large intestine(2/14), and prostate (3/4) cancers.

In addition, increases in BIN1 levels have been observed in differenttypes of cancer or during cancer progression. Gene array analysis of theSW480 colon carcinoma cell line, and their relative lymph nodemetastatic SW620 cells showed statistically significant increase in BIN1transcript level in the metastatic cells SW620 compared to the SW480cells isolated from the primary tumor. In another study in which 22primary human advanced gastric cancer tissues and 8 noncancerous gastrictissues were analyzed by high-density oligonucleotide, the level of BIN1transcript was higher in 40% of patient cancer tissues compare to normalgastric tissues. Other studies measured the expression of the BINprotein in tissues of patients with different cancers using antibodiesagainst the N-terminal or the C-terminal domain of BIN1. The resultsshowed strong expression of BIN1 in malignant lymphoma (in 75% ofcases), in malignant glioma (48%), and in testis cancer (43%). Inaddition, a moderate to strong staining was observed in cancer tissuesof patients with colorectal (in 73% of cases), prostate (100%), ovarian(62%), skin (66%), renal (75%), and lung (46%) cancers. (source: HPRD).

Thus, the bridging integrator 1 (BIN1) gene encodes a nucleocytosolicprotein that was initially identified as a Myc-interacting protein withfeatures of a tumor suppressor. BIN is also known as amphiphysin II,amphiphysin-like, and box dependent MYC interacting protein 1.Alternative splicing of the BIN 1 pre-mRNA transcript results in atleast eleven transcript variants encoding different isoforms. Someisoforms of BIN1 are expressed ubiquitously, while others show a tissuespecific expression. BIN1 isoforms 1-7 are expressed in neurons. Isoform8 is skeletal muscle specific, while isoforms 9 and 10 are ubiquitous.Isoforms that are expressed in the central nervous system may beinvolved in synaptic vesicle endocytosis and may interact with dynamin,synaptojanin, endophilin, and clathrin. Aberrant splice variantsexpressed in tumor cell lines have also been described, which includeisoforms that include exon 12a that is normally spliced into BIN1 mRNAwith other exons (exons 12b-12d) in the central nervous system. Exon 12acan have the following nucleotide sequence

(SEQ ID NO: 2) 5′-CTCCGGAAAG GCCCACCAGT CCCTCCGCCT CCCAAACACACCCCGTCCAA GGAAGTCAAG CAGGAGCAGA TCCTCAGCCT GTTTGAGGAC ACGTTTGTCC CTGAGATCAG CGTGACCACC CCCTCCCAG-3′.Alternatively, the nucleotide sequence shows at least 85, 90, or 95percent identity to SEQ ID NO:2 and such variations may or may notresult in amino acid changes in the expressed protein.

BIN1 is generally considered a tumor suppressor. However BIN1 proteinisoforms containing 12a act as a tumor-promotor. Without exon 12a, BIN1is a tumor suppressor by sequestering myc through its myc-bindingdomain, which is encoded by exons 13 and 14. However, on malignanttransformation, 12a+ BIN1 does not sequester the Myc oncogene, freeingMyc to drive the cells into proliferation. Thus an assay specific for12a+ BIN1 can detect a physiological state in which cancer BIN1predominates and/or is active.

There are at least five isoforms of BIN1 that contain the polypeptideencoded by exon 12a (12a+ BIN1): isoforms 1, 4, 5, 6 (also referred toherein as Ca-1), and the isoform referred to herein as Ca-2. Asdisclosed herein, presence of the Ca-1 and/or Ca-2 isoforms areparticularly indicative of cancer. The disclosed method can thereforeinvolve determining the level of a subset of BIN1 isoforms, includingthe levels of Ca-1, Ca-2, or a combination thereof. Therefore, thedisclosed method can involve determining the Ca-1 and/or Ca-2 isoformlevel in the blood sample.

Isoform Ca-2 differs from isoforms 1, 4, 5, and 6 by the absence of thepolypeptide encoded by exon 13. Therefore, the disclosed method canfurther involve detecting a blood level of BIN1 isoforms that contain apolypeptide encoded by at least both exon 12a and exon 13 (i.e.,12a+/13+ BIN1), thereby excluding the Ca-2 isoform. The ratio or thedifference of all 12a+ BIN isoforms to that of the 12a+/13+ subsetdetermines the level of the Ca-2 (i.e., 12a+/13− BIN1) isoform. Ca-1levels can likewise be specifically determined by, for example,detecting a polypeptide encoded by at least exons 10 and 12a of BIN1.

Reference therefore to determination of Ca-1 and/or Ca-2 isoform levelsas used throughout includes the detection of 12a+ BIN1 (i.e.,polypeptides encoded by exon 12a) and optional detection of 13+ BIN(i.e., polypeptides encoded by exon 13a) and/or 10+ BIN1 (i.e.,polypeptides encoded by exon 10) to isolate and determine levels of Ca-1(10+/12a+ BIN1) and/or Ca-2 (12a+/13− BIN1) isoforms.

Provided herein are methods of diagnosing cancer in a subject. Themethods comprise obtaining a blood sample from the subject and detectinga level of Ca-1 and/or Ca-2 isoforms in the sample. An elevated level ofCa-1 and/or Ca-2 isoforms, particularly levels of the Ca-2 isoform,above a control level indicates that the subject has cancer. Therefore,if the subject has an elevated level of Ca-1 and/or Ca-2 isoforms, themethod can further comprise obtaining a tissue sample (biopsy) from thesubject, e.g., for histological examination, or other analysis for thepurpose of confirming and further defining the cancer. Other steps ofdiagnosis are known to those of skill in the art and include additionallaboratory tests (e.g., additional blood tests, urine tests, or tissueanalysis using the same BIN1 markers or other markers), imaging, and thelike. Blood tests that can be used concurrently or subsequent to theBIN1 analysis include analysis of prostate-specific antigen (PSA),cancer antigen 125 (CA125), calcitonin, alpha fetoprotein (AFP), humanchorionic gonadotropin (HCG), and others. In addition, if the subjecthas elevated level of Ca-1 and/or Ca-2 isoforms, the method can furthercomprises imaging the subject to confirm the presence of cancer.Diagnostic imaging techniques for cancer include X-ray, CT, PET, MRI,and ultrasound.

The disclosed method can involve detecting the level of a subset of the12a+ BIN1 isoforms, including the levels of Ca-1, Ca-2, or a combinationthereof. Therefore, the disclosed method can comprise detecting a levelof 12a+/13− (Ca-2) BIN1 isoform in the sample and comparing it to acontrol level. Therefore, the method can comprise obtaining a bloodsample from the subject, detecting a level of 12a+ BIN1 isoforms in thesample, detecting a level of 12a+/13a+ BIN1 isoforms in the sample,comparing the detected level of 12a+ BIN1 isoforms to the detected levelof 12a+/13a+ BIN1 isoforms to determine an 12a+/13− BIN1 (Ca-2) valuefor the sample, and comparing the 12a+/13− BIN1 (Ca-2) value to one ormore control values. In these methods, an elevated 12a+/13− BIN1 (Ca-2)value indicates the subject has a cancer or a likelihood of cancer suchthat the subject requires additional testing. Therefore, if the subjecthas an elevated 12a+/13− BIN1 (Ca-2) value, the method can furthercomprise obtaining a tissue sample from the subject, e.g., forhistological examination, or other analysis for the purpose ofconfirming and further defining the cancer as described above.

Control levels can be used to establish a threshold value, e.g., suchthat a Ca-1 and/or Ca-2 value greater than the threshold value indicatesthe subject has cancer. This threshold value can be determinedempirically by comparing positive controls (samples from subjects withcancer or a particular type or stage of cancer) and negative controls(samples of subjects without cancer or who have been successfullytreated for cancer). Such controls are optionally age matched or matchedaccording to cancer type or stage. In order to distinguish elevated Ca-1and/or Ca-2 values, the threshold value can be set at least 1, 1.5, 2,2.5, 3, 3.5, 4, 4.5, or 5 standard deviations above the mean negativecontrol value. Other statistical methods can be used to set a thresholdvalue that is within the desired predictive power needed for the assay.For example, the threshold value can be set such that there is nostatistically significant difference between the threshold value and thepositive control values using routine statistical analysis.

As used herein, a negative control level can be determined from adifferent subjects) without cancer, or the same subjects) prior to thediagnosis of cancer. Likewise, a positive control value can bedetermined from one or more subjects with cancer. Alternatively, thepositive control can be based on one or more samples containing knownconcentrations of BIN1 isoform(s), such as recombinant BIN1, as in astandard control.

Optionally, the 12a+ BIN1 polypeptide sequence comprises the amino acidsequence LRKGPPVPPP PKHTPSKEVK QEQILSLFED TFVPEISVTT PSQ (SEQ ID NO:1).Alternatively, the amino acid sequence can be at least 85, 90, or 95percent identical to SEQ ID NO:1. Variations in the sequence can includeamino acid insertions, deletions, or substitutions (including, forexample, 1-5 conservative amino acid substitutions).

Optionally, the cancer is a solid tumor (e.g., a carcinoma, melanoma,sarcoma, lymphoma, or neuroblastoma) or a blood-based cancer (e.g.,leukemia or lymphoma). The cancer can, for example, be a primary canceror a metastatic cancer. The cancer can be selected from the groupconsisting of a a lymphosarcoma, a lymphosarcoma, an oral Sarcoma, asoft tissue sarcoma, or a mast cell tumor. The cancer can be selectedfrom the group consisting of a melanoma, a lymphoma, a myoma, amyosarcoma, a round cell tumor, an adenocarcinoma, a fibrosarcoma, or anadenosarcoma. The cancer can be selected from the group consisting of amyelolipoma, osteosarcoma, hemangiosarcoma, sebaceous cancer, hepaticadenoma, and fibrosarcoma. The cancer can be selected from the groupconsisting of a lung cancer, breast cancer, brain cancer, liver cancer,prostate cancer, colon cancer, gastric cancer, pancreatic cancer, bonecancer, ovarian cancer, uterine cancer, cervical cancer, testicularcancer, bladder cancer, renal cancer, thyroid cancer, and leukemia. Forexample, the cancer can be a lung cancer, colorectal cancer, pancreaticcancer, ovarian cancer, or a thyroid cancer. The lung cancer can be anadenocarcinoma, a squamous cell carcinoma, large cell carcinoma, or asmall cell carcinoma. The lung cancer can also be a mesothelioma.

The blood sample can be, for example, whole blood, plasma, or serum. Ablood sample can be obtained by peripheral vein puncture (venipuncture)or other methods known in the art. The blood sample can be obtained froma subject with cancer, or alternatively, from a subject at risk ofdeveloping cancer. For example, the subject can be at risk of developinglung cancer. Risks associated with lung cancer include smoking exposureto asbestos, personal or family history of lung cancer, or sustainedpassive exposure to smoke.

Optionally, the cancer is stage 0, stage I, stage II, stage III, orstage IV cancer. Classifying a cancer by stage uses numerals 0, I, II,III, and IV to describe the progression of cancer. The stage of a cancerindicates how much the cancer has spread and may take into account sizeand metastasis of the tumor to distant organs. Stages 0, I, and IIcancers are considered early stage tumors. Stages III and IV areconsidered late stage cancers. Stage 0 indicates carcinoma in situ,i.e., an early form of a carcinoma defined by the absence of invasion ofsurrounding tissues. Stage I cancers are localized to one part of thebody. Stage II cancers are locally advanced, as are stage III cancers.Whether a cancer is designated as stage II or stage III thereforediffers according to diagnosis. Stage IV cancers have metastasized orspread to other organs or throughout the body. The provided methods canbe used to diagnose early stage cancers (stages 0, I, and II) as well aslate stage (stages III and IV) cancers.

The provided methods can also be used to differentiate early stage(stage 0, I, or II) from late stage (III or IV) cancer, or to monitorcancer progression. Specifically, blood levels of Ca-1 and/or Ca-2increases in some late stage cancers as compared to early stage orcontrol. Therefore, blood levels of eCa-1 and/or Ca-2 in some 0, I, andII stage cancers is lower than the blood level of Ca-1 and/or Ca-2 inthe corresponding stage III or IV cancers. Blood levels of eCa-1 and/orCa-2 can increase in certain cancers with increased metastasis or withan increased tumor size. Thus, provided are methods of determining astage of progression of a cancer in a subject. The methods compriseselecting a subject with cancer, obtaining a blood sample from thesubject, and determining a blood level of Ca-1 and/or Ca-2 isoforms, ora calculated value thereof, in the sample. The blood level of Ca-1and/or Ca-2 isoforms can be compared to a known value or referencesample or with a previous blood sample from the subject.

The blood level of Ca-1 and/or Ca-2 isoforms or a calculated valuethereof can, for example, be compared to a previous blood sample fromthe subject. A previous blood sample can be a sample from the samesubject isolated at a time prior to the isolation of the most recentblood sample. A higher level of expression as compared to a previousblood sample indicates progression or metastasis of the cancer.Progression or metastasis generally indicates the need for additionaltesting, a change in treatment dosage or frequency, or a more aggressivetreatment (i.e., a new treatment agent). A lower level of expression ascompared to previous blood sample indicates improvement in the cancer.Generally, such an improvement indicates the success of the treatment.In such case, the treatment can be continued or even discontinued if thelevel or calculated value thereof for Ca-1 and/or Ca-2 isoforms issufficiently low.

The blood level of Ca-1 and/or Ca-2 isoforms or calculated value thereofcan, for example, be compared to a known value or a reference sample(s).A lower blood level of Ca-1 and/or Ca-2 isoforms or calculated valuethereof as compared to a known value or a reference sample for a stageIII or IV cancer can indicate the subject has stage 0, I, or II cancer.A higher level of Ca-1 and/or Ca-2 isoforms or calculated value thereofas compared to a known value or a reference sample for a stage 0, I, orII cancer can indicate the subject has stage III or IV cancer.Comparable levels of Ca-1 and/or Ca-2 isoforms or calculated valuethereof to a known value or reference sample for a stage 0, I, II, III,or IV cancer can indicate the subject has a stage 0, I, II, III, or IVcancer, respectively.

As used herein, a known value refers to a value (e.g., blood level ofCa-1 and/or Ca-2 isoforms) obtained from a nondiseased sample, adiseased sample, or a group of samples, which can represent, forexample, an untreated sample, a sample from the same subject at variousstages and/or treatment conditions, or a sample from a different subject(treated or untreated). A known value can, for example, be a valueobtained from a blood sample from the same subject prior to thetreatment of the cancer, wherein the cancer has been assigned adesignated stage (e.g., a stage I cancer). A known value can, forexample, be a value obtained from a blood sample from the same subjectafter treatment of the cancer. A reference sample can, for example,include an untreated subject with stage 0, I, II, III, or IV cancer. Byway of another example, a reference sample can include a treated subjectwith stage 0, I, II, III, or IV cancer. By way of another example, areference sample can be the baseline level of expression in a subjectwith a stage 0, I, II, III, or IV cancer. Reference samples or value caninclude a known value or can be positive or negative control samples(optionally, matched for age, stage of cancer, or type of cancer withthe experimental sample(s)) run in parallel with the experimentalsample.

Ca-1 and/or Ca-2 isoform levels are in some cases higher in youngersubjects than in older subjects. Therefore, in some cases, the subjectof the disclosed methods is at least 35, 40, 45, 50, or 55 years of age.Optionally, the blood level of Ca-1 and/or Ca-2 isoforms and positiveand negative control values are normalized for the age of the subject.

Also provided is a method of determining efficacy of a cancer therapy ina subject based on a change or changes in the Ca-1 and/or Ca-2 bloodlevels or calculated value thereof. The method comprises obtaining afirst blood sample from a subject with cancer prior to treatment with afirst cancer therapy, determining a first Ca-1 and/or Ca-2 value in thefirst blood sample (i.e., as a baseline measurement), obtaining a secondblood sample from a subject with cancer after at least one treatmentwith the first cancer therapy, determining a second Ca-1 and/or Ca-2value in the second blood sample (i.e., as a means of assessing thetreatment effect), and comparing the first value to the second value. Inthis method, a decrease in Ca-1 and/or Ca-2 from the first to the secondblood sample is an indication of effective cancer therapy that can becontinued, e.g., until blood levels reach negative control levels orreduced to a maintenance dosing regimen. However, minimal decrease or anincrease in Ca-1 and/or Ca-2 isoform levels from the first to the secondblood sample is an indication that the cancer therapy is insufficientlyeffective and that a second cancer therapy or an increase in dosingregimen (increased dosage or frequency using the current treatmentagent) for the subject should be selected. A second cancer therapy canalso include administration of multiple chemotherapeutics incombination, surgery, and/or radiation therapy. One of skill in the artcan determine the proper dosages or change in treatment regimen.

BIN1 isoforms without the polypeptide encoded by exon 12a function astumor suppressors, and this activity may be related to the suppressionof indoleamine 2,3-dioxygenase (IDO) (Muller A J, et al., NatureMedicine 2005 11(3):312-319). IDO has been shown to be active inparticular forms of cancer, including lung cancer (Smith C, et al.,Cancer Discovery 2012 2(8):723-735). Cancer therapeutics are beingdeveloped that mimic BIN1 suppression of IDO (Novitskiy S V and Moses HL. Cancer Discovery 2012 2(8):673-5). The disclosed methods maytherefore by used to detect the physiological state in which IDO isactive. Because IDO blocking agents are being developed as cancertherapeutics, the disclosed methods may be used to identify thatparticular subset of cancer patients who will respond to IDO blockingtherapeutics. Among the current clinical trials registered using IDOantagonists, lung cancer is being targeted, and a significant portion oflung cancers are shown herein to have a high blood levels of BIN1 cancerisoform signal.

Therefore, also provided are methods of treating cancer in a subjectthat comprise administering an inhibitor of IDO to the subject having anelevated Ca-1 and/or Ca-2 value. The method involves obtaining a bloodsample from the subject, determining a blood level of Ca-1 and/or Ca-2isoforms or a calculated value thereof, and comparing these levels toone or more control levels or values. In these methods, determination ofelevated blood levels of Ca-1 and/or Ca-2 isoforms or a calculated valuethereof is an indication that the subject has a subset of cancers thatshould be treated with an inhibitor of IDO. Examples of IDO inhibitorsinclude 1-methyl-tryptophan (1-MT), 1-methyl-D-tryptophan, andINCB024360 (InCyte, Wilmington, Del.).

Ca-1 and/or Ca-2 may also be a more specific marker in disease statesthat correspond to an elevated blood level of IDO such as tuberculosis.Therefore, also disclosed are methods of using Ca-1 and/or Ca-2 as adiagnostic and as an assay for evaluating the treatment effectiveness ofIDO-related diseases.

Also provided are methods of detecting the recurrence of cancer in asubject. The methods comprise selecting a subject with a cancer inremission, obtaining a blood sample from the subject, and determining alevel of Ca-1 and/or Ca-2 isoforms or a calculated value thereof in theblood sample. An elevated level of Ca-1 and/or Ca-2 isoforms orcalculated value thereof as compared to a negative control level orvalue indicates that the subject has a recurrence of cancer or is atrisk for a recurrence of cancer. If recurrence or the risk of recurrenceis detected, additional tests or therapy can be performed. Such testsand therapy are described herein and are within the skill in the art.

The level of Ca-1 and/or Ca-2 isoforms can, for example, be determinedby detecting 12a+ BIN1 polypeptide in the biological sample. Optionally,the level of 12a+ BIN polypeptide is determined using an antibody thatis specific for the polypeptide encoded by exon 12a of BIN1 (12a+ BIN1)specific antibody. For example, the antibody can optionally selectivelybind SEQ ID NO:1 (polypeptide encoded by exon 12a) but does not bindisoform 2, which lacks exon 12a. Human BIN1 isoform 2 can have thefollowing amino acid sequence sequence:

MAEMGSKGVT AGKIASNVQK KLTRAQEKVL QKLGKADETKDEQFEQCVQN FNKQLTEGTR LQKDLRTYLA SVKAMHEASKKLNECLQEVY EPDWPGRDEA NKIAENNDLL WMDYHQKLVDQALLTMDTYL GQFPDIKSRI AKRGRKLVDY DSARHHYESLQTAKKKDEAK IAKPVSLLEK AAPQWCQGKL QAHLVAQTNLLRNQAEEELI KAQKVFEEMN VDLQEELPSL WNSRVGFYVNTFQSIAGLEE NFHKEMSKLN QNLNDVLVGL EKQHGSNTFTVKAQPSDNAP AKGNKSPSPP DGSPAATPEI RVNHEPEPAGGATPGATLPK SPSQFEAPGP FSEQASLLDL DFDPLPPVTSPVKAPTPSGQ SIPWDLWEPT ESPAGSLPSG EPSAAEGTFAVSWPSQTAEP GPAQPAEASE VAGGTQPAAG AQEPGETAASEAASSSLPAV VVETFPATVN GTVEGGSGAG RLDLPPGFMFKVQAQHDYTA TDTDELQLKA GDVVLVIPFQ NPEEQDEGWLMGVKESDWNQ HKELEKCRGV FPENFTERVP(SEQ ID NO: 3, Accession No. NP_647594.1).

Therefore, an isolated antibody is disclosed that can selectively bindSEQ ID NO:1 but not bind SEQ ID NO:3. The antibody can be a monoclonalantibody or a recombinant antibody. A monoclonal antibody (9D7 1C1) thatspecifically binds exon 12a is disclosed and described in Example 3. Thecomplementarity determining regions (CDRs) of the 9D7 1C1 antibody'sheavy chain comprises the amino acid sequences SEQ ID NO:10, SEQ IDNO:11, and SEQ ID NO:12. The CDRs of the 9D7 1C1 antibody's light chaincomprise the amino acid sequences SEQ ID NO:13, SEQ ID NO:14, and SEQ IDNO:15. Therefore, the disclosed monoclonal or recombinant antibody thatselectively binds the 12a+ BIN1 polypeptide comprises at least theseCDRs, or CDRs having at least 95% to 99% identity with SEQ ID NO:10, SEQID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.

Examples of analytical techniques useful in determining the expressionof 12a+ BIN1 polypeptide include immunohistochemistry, Western blot,enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (ETA),radioimmunoassay (RIA), protein array, or fluorescent activated cellsorting (FACS). Using a specific antibody against exon 12a BIN1polypeptide, FACS can be used to detect cells expressing exon 12a+ BIN1circulating in blood and/or microparticles and/or tumor cells and/orapoptotic cell fragments expressing 12a+ BIN1 circulating in plasma orserum. These techniques are known by one of skill in the art. See, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, 3^(rd) Ed.,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001).

Immunohistochemical methods may also be used for detecting theexpression levels of 12a+ BIN polypeptide. Thus, antibodies or antisera,such as, polyclonal antisera and monoclonal antibodies specific for 12a+BIN1 polypeptides may be used to assess 12a+ BIN1 polypeptideexpression. The antibodies can be detected by direct labeling of theBIN1 antibodies themselves, for example, with radioactive labels,fluorescent labels, hapten labels such as biotin, or an enzyme such ashorse radish peroxidase or alkaline phosphatase. Alternatively,unlabeled primary antibody is used in conjunction with a labeledsecondary antibody, comprising antisera, polyclonal antisera or amonoclonal antibody that binds the primary antibody. Labeled tertiaryantibodies can be used similarly. Optionally, 12a+ BIN1 polypeptideexpression in a blood sample from a patient may be compared to 12a+ BIN1expression in a blood sample of a normal subject or the same subjectbefore or after cancer.

In certain cases, the level of 12a+ BIN1 isoforms present in a bloodsample may be determined by a Western blot. For example, polypeptidespresent in the whole cell lysate from a blood sample may be separated bySDS-PAGE; the separated polypeptides transferred to a nitrocellulosemembrane; 12a+ BIN1 polypeptide detected by using an antibody orantiserum specific for BIN1 or a specific isoform of 12a+ BIN1. At leastone normalizing polypeptide, for example, CaV 1.2 or a housekeepingpolypeptide such as GAPDH can be detected simultaneously or in paralleland used to normalize the BIN polypeptide expression levels. BIN1expression level may be determined by performing a BIN1immunoprecipitation using an excess of anti-BIN1 antibody (e.g., anantibody specific for 12a+ BIN1 polypeptide). The immunoprecipitation isfollowed by separation of the immunoprecipitate by SDS-PAGE; theseparated polypeptides are transferred to a nitrocellulose membrane; anddetected by staining the gel, e.g., by Coomassie Blue or silver stainingImmunoprecipitation of a control protein such as GAPDH or ubiquitin mayalso be carried out either simultaneously or in parallel. Optionally,the same procedure may be carried out on corresponding normal tissue orfrom a sample from a normal subject.

In certain cases, the level of 12a+ BIN1 isoforms in cells ormicroparticles within human blood can be determined by FACS analysis.FACS is an established method used to detect cells as well ascirculating microparticles. Microparticle analysis by FACS has beensuccessfully used for thrombotic disease diagnosis and prognosis. Incancer, in particular metastasized cancer, tumor cells expressing 12a+BIN can be potentially released into circulation. These cells mayrelease microparticles carrying 12a+ BIN1. Human blood samples can befixed in paraformaldehyde (PFA) followed by labeling the cells and/ormicroparticles with antibody specifically against 12a+ BIN1 polypeptide.The antibodies can be detected by direct labeling of the BIN1 antibodieswith fluorescent labels or unlabeled primary antibody used inconjunction with a labeled secondary antibody, comprising antisera,polyclonal antisera, or a monoclonal antibody specific for the primaryantibody. Fluorescently labeled 12a+ BIN1 positive cells and/ormicroparticles can be sorted out by FACS analysis. Optionally, 12a+ BIN1polypeptide expression in a blood sample from a patient may be comparedto 12a+ BIN1 polypeptide expression in a blood sample in a normalsubject or the same subject before or after cancer. Similarly, a mobilesold support like fluorescent beads with bound antibody (e.g., antibodyselective for 12a+ BIN1) can be used in FACS analysis, wherein beads ofdiffering fluorescence are used to correlate with different boundantibodies.

Optionally, the level of 12a+ BIN1 expression can be determined bydetecting a BIN1 nucleic acid comprising exon 12a (e.g., exon 12a+ BIN1mRNA), or fragment thereof, in the sample. Examples of analyticaltechniques useful in determining the expression of exon 12a+ BIN1 mRNAinclude reverse transcription-polymerase chain reaction (RT-PCR),quantitative real time-PCR (qRT-PCR), one step PCR, RNase protectionassay, primer extension assay, microarray analysis, gene chip, in situhybridization, and Northern blot.

When RT-PCR is used to determine exon 12a+ BIN1 mRNA expression, mRNAcan be isolated from the sample. Optionally, RNA is isolated from bloodor plasma of the subject. Normal blood or plasma of another subject canbe a control. A normal blood or plasma sample from the same subjectbefore cancer or after cancer is successfully treated can be a control.

General methods for mRNA extraction are well known in the art and aredisclosed in standard textbooks of molecular biology, including Ausubelet al., Current Protocols of Molecular Biology, John Wiley and Sons(1997). Methods for RNA extraction from paraffin embedded tissues aredisclosed, for example, in Rupp and Locker, Lab Invest. 56:A67 (1987),and De Andrés et al., BioTechniques 18:42044 (1995). Optionally, RNAisolation can be performed using a purification kit, buffer set andprotease from commercial manufacturers according to the manufacturer'sinstructions. For example, total RNA can be isolated using QiagenRNeasy® mini-columns (Hilden, Del.). Other commercially available RNAisolation kits include MasterPure® Complete DNA and RNA Purification Kit(EPICENTRE®, Madison, Wis.), and Paraffin Block RNA Isolation Kit®(Ambion, Inc., Austin, Tex.). Total RNA from tissue samples can beisolated using RNA Stat-60® (Tel-Test, Friendswood, Tex.). RNA preparedfrom a biological sample can be isolated, for example, by cesiumchloride density gradient centrifugation.

The RNA template can be transcribed into cDNA, followed by itsexponential amplification in a PCR reaction. One or more of a number ofreverse transcriptases may be used, including, but not limited to, AvianMyeloblastosis Virus Reverse Transcriptase (AMV-RT), Moloney MurineLeukemia Virus Reverse Transcriptase (MMLV-RT), reverse transcriptasefrom human T-cell leukemia virus type I (HTLV-I), bovine leukemia virus(BLV), Rous sarcoma virus (RSV), human immunodeficiency virus (HIV) andThermus thermophilus (Tth). The reverse transcription step is typicallyprimed using specific primers, random hexamers, or oligo-dT primers,depending on the circumstances and the goal of RT-PCR. For example,extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit(Perkin Elmer; Waltham, Mass.), following the manufacturer'sinstructions. The derived cDNA can then be used as a template in thesubsequent PCR reaction.

Although the PCR step can use a variety of thermostable DNA-dependentDNA polymerases, typically employed is the Taq DNA polymerase, which hasa 5′-3′ nuclease activity but lacks a 3′-5′ proofreading endonucleaseactivity. Thus, TaqMan® PCR typically utilizes the 5′-nuclease activityof Taq or Tth polymerase to hydrolyze a hybridization probe bound to itstarget amplicon, but any enzyme with equivalent 5′ nuclease activity canbe used. Two oligonucleotide primers are used to generate an amplicontypical of a PCR reaction. A third oligonucleotide, or probe, isdesigned to detect nucleotide sequence located between the two PCRprimers. The probe is non-extendible by Taq DNA polymerase enzyme and islabeled with a reporter fluorescent dye and a quencher fluorescent dye.Any laser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

RT-PCR can be performed using commercially available equipment, such as,for example, ABI PRISM 7700TM Sequence Detection System®(Perkin-Elmer-Applied Biosystems; Foster City, Calif.), or Lightcycler®(Roche Molecular Biochemicals; Mannheim, Del.). Optionally, the 5′nuclease procedure is run on a real-time quantitative PCR device. Such asystem can comprise a thermocycler, laser, charge-coupled device (CCD),camera and computer. The system amplifies samples in a 96-well format ona thermocycler. During amplification, laser-induced fluorescent signalis collected in real-time through fiber optics cables for all 96 wells,and detected at the CCD. The system includes software for running theinstrument and for analyzing the data.

5′-Nuclease assay data are initially expressed as a threshold cycle(Ct). Fluorescence values are recorded during every cycle and representthe amount of product amplified to that point in the amplificationreaction. The point when the fluorescent signal is first recorded asstatistically significant is the threshold cycle (Ct).

To minimize errors and the effect of sample-to-sample variation, RT-PCRis optionally performed using an internal standard. The ideal internalstandard is expressed at a constant level among different tissues, andis unaffected by the experimental treatment. RNAs most frequently usedto normalize patterns of gene expression are mRNAs for the housekeepinggenes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and β-actin.

A variation of the RT-PCR technique is the real time quantitative PCR,which measures PCR product accumulation through a dual-labeledfluorogenic probe. Real time PCR is compatible both with quantitativecompetitive PCR, where internal competitor for each target sequence isused for normalization, and with quantitative comparative PCR using anormalization gene contained within the sample, or a housekeeping genefor RT-PCR.

To correct for (normalize away) both differences in the amount of RNAassayed and variability in the quality of the RNA used the assay canoptionally incorporate analysis of the expression of certain referencegenes (or “normalizing genes”), including well known housekeeping genes,such as GAPDH, HPRT1, ubiquitin, etc.

Alternatively, normalization can be based on the mean or median signalof all of the assayed genes or a large subset thereof (often referred toas a “global normalization” approach). On a gene-by-gene basis, measurednormalized amount of a subject tissue mRNA may be compared to the amountfound in a corresponding normal tissue.

For example, primers and probes (e.g., for use in PCRamplification-based methods) can be designed based upon an exon sequenceto be amplified. Accordingly, the primer/probe design can includedetermining a target exon sequence within the gene of interest (e.g.,exon 12a of BIN1). This can be done by publicly available software, suchas the DNA BLAST software developed by Kent, W. J., Genome Res.12(4):656-64 (2002), or by the BLAST software including its variations.Subsequent steps follow well established methods of PCR primer and probedesign.

In order to avoid non-specific signals, repetitive sequences within thetarget sequence of the gene can be optionally masked when designing theprimers and probes. The masked sequences can then be used to designprimer and probe sequences using any commercially or otherwise publiclyavailable primer/probe design packages, such as Primer Express (AppliedBiosystems; Carlsbad, Calif.); MGB assay-by-design (Applied Biosystems;Carlsbad, Calif.).

Factors to be considered in PCR primer design can include primer length,melting temperature (Tm), G/C content, specificity, complementary primersequences, and 3′-end sequence. PCR primers can optionally be 17-30bases in length, and contain about 20-80% G+C bases, (e.g., about 50-60%G+C bases). Tms are between 50° C. and 80° C., e.g. about 50° C. to 65°C.

Microarray technology may be used to detect differential expression ofexon 12a+BIN1 in a subject's blood sample and normal or control bloodsample. In this method, polynucleotide sequences of interest (includingcDNAs and oligonucleotides) are plated, or arrayed, on a microchipsubstrate. The arrayed sequences are then hybridized with specific DNAprobes from blood samples of interest. Similar to the RT-PCR method, thesource of mRNA is optionally total RNA isolated from subject's bloodsample, and optionally corresponding normal or control blood sample.

Fluorescently labeled cDNA probes can be generated through incorporationof fluorescent nucleotides by reverse transcription of RNA extractedfrom tissues of interest. Labeled cDNA probes applied to the chiphybridize with specificity to each spot of DNA on the array. Afterstringent washing to remove non-specifically bound probes, the chip isscanned by confocal laser microscopy or by another detection method,such as a CCD camera. Quantitation of hybridization of each arrayedelement can be used for assessment of corresponding mRNA abundance.

With dual color fluorescence, separately labeled cDNA probes generatedfrom two sources of RNA are hybridized pair wise to the array. Therelative abundance of the transcripts from the two sources correspondingto each specified gene is thus determined simultaneously. Microarraymethods have been shown to have the sensitivity to detect raretranscripts, which are expressed at a few copies per cell, and toreproducibly detect at least approximately two-fold differences in theexpression levels (Schena et al., Proc. Natl. Acad. Sci. USA93(2):106-149 (1996)).

The arrayed oligonucleotides may include oligonucleotides whichhybridize to a specific region of the exon 12a+ BIN1 nucleic acid. Incertain embodiments, multiple copies of a first oligonucleotide whichspecifically hybridizes to a first region of the exon 12a+ BIN1 nucleicacid are arrayed. In certain embodiments, multiple copies of first and asecond oligonucleotide which specifically hybridize to a first and asecond region of the exon 12a+ BIN1 nucleic acid, respectively, arearrayed, and so on. In certain embodiments, the exon 12a+ BIN1 nucleicacid expression level is determined by mean values of the signal fromeach of these oligonucleotides. The array may also includeoligonucleotides which specifically hybridize to nucleic acid of anormalizing gene, such as a housekeeping gene or other genes known notto be significantly differentially expressed in diseased versus normaltissue, for example, CaV 1.2.

Optionally, the BIN1 polypeptide, nucleic acid, or fragments of saidpolypeptides or nucleic acids detected is human. Optionally, BIN1polypeptide, nucleic acid, or fragments of said polypeptides or nucleicacids detected is non-human mammal (e.g., rodent, porcine, bovine,equine, canine, or feline).

There are a variety of BIN1 sequences that are disclosed on Genbank, andthese sequences and others are herein incorporated by reference in theirentireties as are individual subsequences or fragments containedtherein. As used herein, BIN1 includes homologs, variants, and isoformsthereof.

The nucleotide and amino acid sequences of BIN1 isoforms 1-10 can befound at GenBank Accession Nos. NM_(—)139343.2 and NP_(—)647593.1 forisoform 1; NM_(—)139344.2 and NP_(—)647594.1 for isoform 2;NM_(—)139345.2 and NP_(—)647595.1 for isoform 3; NM_(—)139346.2 andNP_(—)647596.1 for isoform 4; NM_(—)139347.2 and NP_(—)647597.1 forisoform 5; NM_(—)139348.2 and NP_(—)647598.1 for isoform 6;NM_(—)139349.2 and NP_(—)647599.1 for isoform 7; NM_(—)004305.3 andNP_(—)04296.1 for isoform 8; NM_(—)139350.2 and NP_(—)647600.1 forisoform 9; and NM_(—)139351.2 and NP_(—)647601.1 for isoform 10. Twoother reported exon 12a+ BIN1 tumor isoforms include BIN1+12a found atGenBank Accession Nos. AF068918.1 and AAC23751.1 for nucleotide andamino acid sequences, respectively, and BIN1-10+12a found at GenBankAccession Nos. AF068917.1 and AAC23750.1 for nucleotide and amino acidsequence, respectively. The nucleotide and amino acid sequence of exon12a is given by SEQ ID NO:2 and SEQ ID NO:1, respectively.

Thus, provided are the nucleotide sequences of BIN1 comprising exon 12a+(SEQ ID NO:2) comprising a nucleotide sequence at least about 70%, 75%,80%, 85%, 90%, 95%, 98%, 99% or more identical to the nucleotidesequences of the aforementioned GenBank Accession Numbers. Also providedare amino acid sequences of the BIN1 polypeptide comprising the encodedamino acid sequence of exon 12a+ (SEQ ID NO:1) comprising an amino acidsequence at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or moreidentical to the sequences of the aforementioned GenBank AccessionNumbers.

Antibodies that bind the polypeptides described above, including 12a+BIN1, or polypeptide fragments thereof, can be used to detected 12a+BIN1 isoforms in a biological sample. For example, the polypeptidesdescribed above can be used to produce antibodies to 12a+ BIN1.

As used herein, the term antibody encompasses, but is not limited to,whole immunoglobulin (i.e., an intact antibody) of any class. Chimericantibodies and hybrid antibodies, with dual or multiple antigen orepitope specificities, and fragments, such as F(ab′)2, Fab′, Fab and thelike, including hybrid fragments are useful herein. Thus, fragments ofthe antibodies that retain the ability to bind their specific antigensare provided and are useful in the methods taught here. For example,fragments of antibodies which maintain binding activity to 12a+ BIN1expressed in cancers are included within the meaning of the termantibody or fragment thereof. Such antibodies and fragments can be madeby techniques known in the art and can be screened for specificity andactivity according to general methods for producing antibodies andscreening antibodies for specificity and activity (See Harlow and Lane.Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, NewYork (1988)).

Also useful in the methods herein are conjugates of antibody fragmentsand antigen binding proteins (single chain antibodies) as described, forexample, in U.S. Pat. No. 4,704,692, the contents of which are herebyincorporated by reference in their entirety.

Optionally, the antibody is a monoclonal antibody. The term monoclonalantibody as used herein refers to an antibody from a substantiallyhomogeneous population of antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Monoclonalantibodies may be prepared using hybridoma methods, such as thosedescribed by Kohler and Milstein, Nature, 256:495 (1975) or Harlow andLane, Antibodies, A Laboratory Manual. Cold Spring Harbor Publications,New York (1988). In a hybridoma method, a mouse or other appropriatehost animal is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro. The immunizing agent can be 12a+BIN expressed in cancer or an immunogenic fragment thereof.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies can be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of murine antibodies). The DNA also may be modified, for example,by substituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody provided herein, or can be substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for 12a+ BIN1 expressed in cancer and anotherantigen-combining site having specificity for a different antigen.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348, U.S. Pat. No. 4,342,566,and Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York, (1988). Papain digestion of antibodies typicallyproduces two identical antigen binding fragments, called Fab fragments,each with a single antigen binding site, and a residual Fc fragment.Pepsin treatment yields a fragment, called the F(ab′)2 fragment that hastwo antigen combining sites and is still capable of cross-linkingantigen.

The Fab fragments produced in the antibody digestion can also containthe constant domains of the light chain and the first constant domain ofthe heavy chain. Fab′ fragments differ from Fab fragments by theaddition of a few residues at the carboxy terminus of the heavy chaindomain including one or more cysteines from the antibody hinge region.The F(ab′)2 fragment is a bivalent fragment comprising two Fab′fragments linked by a disulfide bridge at the hinge region. Fab′-SH isthe designation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group.

Further provided herein is a humanized or human version of the antibody.Humanized and human antibodies can be made using methods known to askilled artesian; for example, the human antibody can be produced usinga germ-line mutant animal or by a phage display library.

Antibodies can also be generated in other species and humanized foradministration to humans. Alternatively, fully human antibodies can alsobe made by immunizing a mouse or other species capable of making a fullyhuman antibody (e.g., mice genetically modified to produce humanantibodies) and screening clones that bind exon 12a+ BIN1 expressed incancer. See, e.g., Lonberg and Huszar, Int. Rev. Immunol. 13:65-93,(1995), which is incorporated herein by reference in its entirety formethods of producing fully human antibodies. As used herein, the termhumanized and human in relation to antibodies, relate to any antibodywhich is expected to elicit a therapeutically tolerable weak immunogenicresponse in a human subject. Thus, the terms include fully humanized orfully human as well as partially humanized or partially human.

Kits containing one or more of the disclosed antibodies are alsoprovided for detecting Ca-1 and/or Ca-2 isoform levels. The kit cancontain an assay system for detecting 12a+ BIN1 polypeptides and anassay system for detecting 12a+/13+ BIN1 polypeptides. For example, thekit can contain a first assay system for detecting 12a+ BIN isoformsthat comprises an antibody that selectively binds 12a+ BIN1, and anantibody that selectively binds multiple human BIN1 isoforms. The assaymay also be a sandwich assay, wherein one of these two antibodies isimmobilized on a solid surface. The kit can contain a second assaysystem for detecting 12a+/13+ BIN1 polypeptides that comprises anantibody that selectively binds 13+ BIN1, and an antibody thatselectively binds 12a+ of BIN1. The assay may also be a sandwich assay,wherein one of these two antibodies is immobilized on a solid surface.The solid support can include a plate, array, chip or bead. Optionallythe antibodies of the kit are labeled. The kit optionally includes oneor more secondary and/or tertiary antibodies (optionally labeled),containers for the antibodies, and/or regents for detection of thelabels. The assay system optionally includes one or more solid supportswith the selected antibody or antibodies bound thereto.

As used throughout, subject can be a vertebrate, more specifically amammal (e.g., a human, horse, cat, dog, cow, pig, sheep, goat, mouse,rabbit, rat, and guinea pig), birds, reptiles, amphibians, fish, and anyother animal. The term does not denote a particular age or sex. Thus,adult and newborn subjects, whether male or female, are intended to becovered. As used herein, patient or subject may be used interchangeablyand can refer to a subject with a disease or disorder (e.g., cancer).The term patient or subject includes human and veterinary subjects.

As used herein the terms treatment, treat, or treating refers to amethod of reducing the effects of a disease or condition or symptom ofthe disease or condition. Thus in the disclosed method, treatment canrefer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%reduction in the severity of an established disease or condition orsymptom of the disease or condition. For example, a method for treatinga disease is considered to be a treatment if there is a 10% reduction inone or more symptoms of the disease in a subject as compared to acontrol. Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, or any percent reduction in between 10% and 100% ascompared to native or control levels. It is understood that treatmentdoes not necessarily refer to a cure or complete ablation of thedisease, condition, or symptoms of the disease or condition.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a method is disclosed and discussed and a numberof modifications that can be made to a number of molecules including themethod are discussed, each and every combination and permutation of themethod, and the modifications that are possible are specificallycontemplated unless specifically indicated to the contrary. Likewise,any subset or combination of these is also specifically contemplated anddisclosed. This concept applies to all aspects of this disclosureincluding, but not limited to, steps in methods of using the disclosedcompositions. Thus, if there are a variety of additional steps that canbe performed it is understood that each of these additional steps can beperformed with any specific method steps or combination of method stepsof the disclosed methods, and that each such combination or subset ofcombinations is specifically contemplated and should be considereddisclosed.

Publications cited herein and the material for which they are cited arehereby specifically incorporated by reference in their entireties.

EXAMPLES Example 1 Canine Study of BIN1 in Blood Samples Methods

Canine Selection and Serum Acquisition.

Venous blood samples were obtained from 31 dogs with a definitediagnosis of carcinoma and seven healthy dogs as controls. Two sampleswere excluded due to high muscle contaminant (creatinine kinase >1000IU/L) and another two samples were excluded due to incomplete clinicaldata. The remaining 27 samples had their cancer staged (I-IV), accordingto standardized clinical (not biopsy based) staging guidelines for eachrespective cancer. The cancers were a mix between solid and blood-basedtumors, including lymphoma, sarcoma, adenosarcoma, and undifferentiatedtumor.

Each dog was restrained in a sternal recumbancy. 5 mls of venous bloodwas collected into a 7.0 ml glass EDTA tube from the jugular vein usinga 12.0 ml syringe with a 21 gauge-1 inch needle. After mixed with EDTA,the blood was then centrifuged at 4,000 rpm for 20 minutes at 4° C. Thesupernatant serum was collected into a 1.7 ml Eppendorf tube and storedin −80° C. freezer for later analysis.

Detection of Serum BIN1 Protein by Capture ELISA.

Round bottomed 96-well plates were coated at 4° C. for 16 hours withmouse anti BIN1 (clone 99D, Sigma, 1/1000) (Sigma; St. Louis, Mo.)diluted in 0.1 M sodium carbonate buffer, pH 9.0. The plates were washedthree times with tris-buffered saline tween-20™ (TBST) to remove unboundantibody and blocked for 1 hour at room temperature with 1% bovine serumalbumin (BSA) in TBST (blocking buffer). 100 μl of each serum sample wasadded, in duplicate, and plates were incubated overnight at 4° C. withorbital rotation. The samples were then aspirated and plates were washedtwice quickly, followed by three times for 5 minutes with TBST. Goatanti-BIN1 (1/1000 in blocking buffer) (Everest Biotech; Oxfordshire,United Kingdom) was then applied as a detection antibody, and the plateswere incubated for 2 hours at room temperature with rotation. Thedetection antibody was then aspirated and the plates were washed twicequickly, followed by three times for 5 minutes with TBST. The plateswere subsequently incubated for 1 hour at room temperature withHRP-conjugated donkey anti-goat IgG (1/2000 in blocking buffer) (Abcam;Cambridge, Mass.) before two quick washes and three 5 minute washes withTBST. TMB substrate was added and plates were incubated in the dark for1 hour before reaction termination with 1 N hydrochloric acid (HCL).Following the reaction termination, the plates were read using theELx800 BioTek microplate spectrophotometer (BioTek; Winooski, Vt.), andOD values were determined at 405 nm. All values were normalized to thatof a two year old, 9 kilogram healthy dog.

Results

A canine study was undertaken to determine the correlation between serumBIN1 levels and clinically assessed cancer stage. For this study,twenty-seven dogs with a definite diagnosis of carcinoma were studied.Serum was obtained from the animals, and assayed for BIN1 content byELISA. The capture antibody in the ELISA test was a commerciallyavailable monoclonal BIN1 antibody against the region encoded by BIN1exon 13 (clone 99D, sigma). As indicated in FIG. 1, dogs with limitedcancer (Stage I) has significantly less serum BIN1 that dogs withadvanced cancer (Stage III and IV). Of note, the dogs in this cohort didnot differ significantly between weight, age, or creatininephospho-kinase (indication of muscle sampling).

This proof of principle study is supportive of BIN as a blood availablecancer diagnostic tool. Elevation of BIN1 in the serum fraction ofvenous blood significantly predicts stage III or IV status of carcinomain canines. BIN1 could be a quantitative blood biomarker of metastaticcancer in human.

Example 2 Sequence Analysis of 9D7 1C1 Monoclonal Antibody Materials andMethods

Total RNA Extraction.

Total RNA was extracted from hybridomas using Qiagen kit.

First-Round RT-PCR.

QIAGEN® OneStep RT-PCR Kit (Cat No. 210210) was used. RT-PCR wasperformed with primer sets specific for the heavy and light chains. Foreach RNA sample, 12 individual heavy chain and 11 light chain RT-PCRreactions were set up using degenerate forward primer mixtures coveringthe leader sequences of variable regions. Reverse primers are located inthe constant regions of heavy and light chains. No restriction siteswere engineered into the primers. The reaction setup contained 5.0 μl5×QIAGEN® OneStep RT-PCR Buffer, 0.8 μl dNTP Mix (containing 10 mM ofeach dNTP), 0.5 μl Primer set, 0.8 μl QIAGEN® OneStep RT-PCR Enzyme Mix,2.0 μl Template RNA, and RNase-free water to 20.0 μl. The PCR conditionswere 50° C., 30 min, 95° C., 15 min, 20 cycles of (94° C., 25 sec; 54°C., 30 sec; and 72° C., 30 sec), followed by a final extension at 72°C., 10 min.

Second-Round Semi-Nested PCR.

The RT-PCR products from the first-round reactions were furtheramplified in the second-round PCR. 12 individual heavy chain and 11light chain RT-PCR reactions were set up using semi-nested primer setsspecific for antibody variable regions. The reaction setup contained 10μl 2×PCR mix, 2 μl primer set, and 8 μl of the first round product. ThePCR conditions were 95° C., 5 min, 25 cycles of (95° C., 25 sec; 57° C.,30 sec; and 68° C., 30 sec), followed by a final extension at 68° C., 10min.

After PCR was finished, PCR reaction samples were run onto agarose gelto visualize DNA fragments amplified. The correct antibody variableregion DNA fragments should have a size between 400-500 base pair.

PCR positive bands were TOPO cloned. The TOPO clones were PCR-amplified,followed by gel electrophoresis and recovery from agarose gel.Approximately 24 clones were then sequenced, and CDR analysis wasperformed using these sequence data.

Results

After sequencing cloned DNA fragments, several mouse antibody heavy andlight chains were identified. Antibody CDR analysis identified one heavychain and two light chains. A summary of the sequencing results is shownin Table 2.

TABLE 2 Summary of Antibody Sequence Results Type #Sequencing result summary Heavy chain H1 Heavy chain Heavy chain H8Not an antibody gene Heavy chain H9 Not an antibody gene Light chain L2Not an antibody gene Light chain L3 Not an antibody gene Light chain L4Not an antibody gene Light chain L5 Not an antibody gene Light chain L6Light chain Light chain L7 Light chain (distinct from L6) ---CDR1-->   <--CDR2->   <--CDR3-- MHC299H1_1__M13RGFNIKDYY....__IDPENGNT..__VRGEDYGGYAMDY MHC299H1_2__M13RGFNIKDYY....__IDPENGNT..__VRGEDYGGYAMDY MHC299H1_4__M13RGFNIKDYY....__IDPENGNT..__VRGEDYGGYAMDY MHC299H1_5__M13RGFNIKDYY....__IDPENGNT..__VRGEDYGGYAMDY MHC299L6_1__M13RKSLLHSNGNTY.__RMS.......__MQHLEFPFT MHC299L6_2__M13RKSLLHSNGNTY.__RMS.......__MQHLEFPFT MHC299L6_3__M13RKSLLHSNGNTY.__RMS.......__MQHLEFPFT MHC299L6_5__M13RKSLLHSNGNTY.__RMS.......__MQHLEFPFT MHC299L7_2__M13RQDVSTA......__WAS.......__QQHYSTPFT MHC299L7_3__M13RQDVSTA......__WAS.......__QQHYSTPFT MHC299L7_4__M13RQDVSTA......__WAS.......__QQHYSTPFT

The following are the sequences listed in Table 2:

(SEQ ID NO: 10) GFNIKDYY, (SEQ ID NO: 11) IDPENGNT,  (SEQ ID NO: 12)VRGEDYGGYAMDY, (SEQ ID NO: 13) KSLLHSNGNTY, (SEQ ID NO: 14) MQHLEFPFT,(SEQ ID NO: 15) QDVSTA,  and (SEQ ID NO: 16) QQHYSTPFT.

Variable VH Region Sequences Amino Acid Sequence in FASTA format(MHC299H1.1\;M13R): (SEQ ID NO: 4)EVQLQQSGAELVRPGALVKLSCKASGFNIKDYYVYWVKQRPEQGLEWIGWIDPENGNTIYDPEFQAKASITADTSSNTAYLQLSSLTSEGTAVYYCVRGEDYGGYAMDYWGQGTSVTVSS. Nucleotide Sequence in FASTA format(MHC299H1.1\;M13R): (SEQ ID NO: 5)GAGGTCCAGCTGCAGCAGTCTGGGGCTGAGCTTGTGAGGCCAGGGGCCTTAGTCAAGTTGTCCTGCAAAGCTTCTGGCTTCAACATTAAAGACTACTATGTGTATTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAGAATGGTAATACTATATATGACCCGGAGTTCCAGGCCAAGGCCAGTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGGCACTGCCGTCTATTACTGTGTTAGAGGGGAGGATTACGGGGGCTATGCTATGGACTACTGGGGTCAAGGAACCT CAGTCACCGTCTCCTCA.Variable VL Region Sequences Amino Acid Sequence in FASTA format (MHC299L6.3\;M13R): (SEQ ID NO: 6)DIVVTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLSWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLE FPFTFGSGTKLEIK.Nucleotide Sequence in FASTA format (MHC299L6.3\;M13R): (SEQ ID NO: 7)GATATTGTGGTGACTCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCATAGTAATGGCAACACTTACTTGTCTTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATTTATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATTTCCCTTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAC.Variable VL Region Sequences Amino Acid Sequence in FASTA format (MHC299L7.2\;M13R): (SEQ ID NO: 8)DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQQHYSTPFTF GSGTKLEIK.Nucleotide Sequence in FASTA format  (MHC299L7.2\;M13R): (SEQ ID NO: 9)GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGTATCAACAAAAACCAGGGCAATCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTATACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCACTTTATTACTGTCAGCAACATTATAGCACTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAC.

Example 3 BIN1 Detection in Blood Samples from Human Cancer Patients

The Bridging integrator 1 (BIN1) gene encodes several isoforms of anucleocytoplasmic protein. Twenty exons in the BIN gene arealternatively spliced to give rise to at least ten BIN1 isoforms (FIG.3), with ubiquitous or tissue-specific expression. Two BIN1 isoformsthat contain exon 12a have been described as tumor isoforms (PrendergastG C, et al., Biochim Biophys Acta. 2009 1795(1):25-36): one includesexon 13 (accession number: AF068917.1) with sequence similarity toreported isoform 6 (NM_(—)139348) and is referred as BIN1 isoform 6 inthe literature and BIN1 Ca-1 herein, and the other one does not includeexon 13 and is referred as BIN1 Ca-2 herein and represents a new cancerisoform (FIG. 3).

This study aims at characterizing BIN1 cancer isoforms containing exon12a as circulating cancer biomarkers, by employing combinations ofantibodies of different specificities to indirectly measuring the levelof these isoforms detectable in blood samples from normal and cancer inhumans.

Antibody sandwich assay combinations (Table 3) were designed to detectisoforms that contain polypeptides encoded by both exon 12a and exon 13(Assay #1) or detect isoforms containing polypeptides encoded by exon12a and ubiquitously expressed exon 11 (Assay #2) which is designed tocapture 12a+ BIN1 with and without exon 13. The existence of 12a+/13−BIN1 was detected in the context of the presence of the proto-oncogenesplicing factor SF2/ASF (Karni R, et al., Nature Struct Mol Biology.2007 14(3):185-193). This 12a+/13− BIN1 isoform is referred to herein asCa-2 (FIG. 3).

Materials and Methods

Antibodies.

To measure the levels of BIN1 cancer isoforms in blood samples, a set offour BIN1 antibodies was used in enzyme-linked immunosorbent assay(ELISA). The detection of all BIN1 isoforms was performed using ananti-BIN1 goat polyclonal antibody from Everest (cat# EB08724), namedp11 in this study, which specifically recognizes the polypeptide encodedby exon 11, present in all BIN1 isoforms. The detection of the subset ofBIN1 isoforms that contain the polypeptide encoded by exon 13 wasperformed using an anti-BIN1 mouse monoclonal antibody from Sigma (cat#B9428), named m13 in this study. For the detection of the subset of BIN1isoforms that contain the polypeptide encoded by exon 12a, twocustom-made antibodies were used: an anti-BIN1 rabbit polyclonalantibody (#A5299) named p12a in this study, and an anti-BIN1 mousemonoclonal antibody (#9D71C1), named m12a in this study.

Assays and Tests.

Detection of serum BIN1 protein by capture sandwich ELISA. Antibodycombinations are listed in Table 3. Round bottomed 96-well plates werecoated at 4° C. for 16 hours with capture antibody (approximately 5μg/ml) diluted in 0.1 M sodium carbonate buffer, pH 9.0. The plates arewashed three times with tris-buffered saline TWEEN-20 (TBST) to removeunbound antibody and blocked for 1 hour at room temperature with 1%bovine serum albumin (BSA) in TBST (blocking buffer). 50 μl of standards(recombinant BIN1 proteins) and each serum sample was added, induplicate, and plates were incubated overnight at 4° C. with rotation.The samples were then aspirated and plates were washed twice quickly andthree times for 5 minutes with TBST. Primary detection antibody (5 μg/min blocking buffer) was then applied as a detection antibody, and theplates were incubated for 1 hour at room temperature with rotation. Thedetection antibody was then aspirated and the plates were washed twicequickly, followed by three times for 5 minutes with TBST. The plateswere subsequently incubated for 1 hour at room temperature withHRP-conjugated secondary antibody (1/2000 in blocking buffer) before twoquick washes and three 5 minute washes with TBST.3,3′,5,5′-Tetramethylbenzidine (TMB) substrate was added and plates wereincubated in the dark for 1 hour before reaction termination with 1 Nhydrochloric acid (HCL). Following the reaction termination, the plateswere read using an ELx800 microplate spectrophotometer (BIOTEK,Winooski, Vt.) and optical density (OD) values were determined at 405nm. A standard curve was generated from the OD values of the proteinstandards of known protein concentration. BIN1 concentrations of eachsample were then derived from the standard curve.

Two different combinations of these antibodies were used and defined twodistinct assays (Table 3). In Assay #1, m13 was used for capture andp12a was used for detection to measure the levels of four BIN1 isoformsincluding the cancer isoform Ca-1. In Assay #2, m12 was used for captureand p11 was used for detection to measure the levels of five BIN1isoforms including the two cancer isoforms Ca-1 and Ca-2.

TABLE 3 BIN1 antibodies used in different assays and BIN1 isoformsdetected. (Ca-1 and Ca-2 represent BIN1 cancer isoforms) CaptureDetection BIN1 isoforms detected Assay #1 m13 p12a 1, 4, 5, Ca-1 Assay#2 m12a p11 1, 4, 5, Ca-1, Ca-2 For Assay #2 the secondary HRPconjugated antibody was donkey anti-goat IgG (Abcam). For Assay #1 thesecondary HRP conjugated antibody was goat anti-rabbit IgG (Abcam). Toevaluate the levels of BIN1 new cancer isoform Ca-2, the ratio Assay#2/Assay #1 was used and termed BIN1 Cancer Test #1.

Human Samples.

Commercial vendors with human clinical sample repositories, collectedunder IRB and including relevant clinical data were identified.Forty-two blood samples were obtained (Conversant, Asterand) fromunique, clinically normal subjects ranging from 25 to 79. Fifty humanblood samples; ten for each of lung, pancreas, colorectal, ovarian andthyroid cancer, were obtained (Innovative Research and Conversant). Theages of the cancer patients ranged from 18 to 84. Blood samples wereobtained from lung cancer patients with stage I and IV disease, frompancreas cancer patients with cancer stage III and IV disease, fromcolorectal cancer patients with stage II, III and IV disease, andovarian and thyroid cancer patients with all disease stages (I-IV).

Results

Standard Curves.

As a positive control for the ELISA assays used in this study, BIN1recombinant protein (Ca-1) was overexpressed in human HEK-293 cells andthe total lysate was analyzed using Assay #1 (m13-p12a) and Assay #2(m12-p11). The two standard curves for Assay #1 and Assay #2 arerepresented in FIG. 4A and FIG. 4B, respectively. In both Assay #1 andAssay #2, an increase in the amount of BIN1 recombinant proteincorrelates with an increase of signal detected using BIN1 antibodies.

FIG. 5 demonstrates the relationship between the two cancer isoforms,Ca-1 and Ca-2. Many samples give a high signal with Assay #1 (x-axis),both normals and cancer. Combining this with the signal for Assay #2results in a much better cancer specificity. Ten cancer samples(triangles) with Assay #2 levels above 10 also had Assay #1 levels above10. As discussed further below, looking at these samples by age is alsoinformative, the higher signal in Normals occurs in samples from youngersubjects. This graph demonstrates that both assays show increased signalin cancer, indicating that both Ca-1 and Ca-2 are important. However,samples are analyzed below using a ratio of Assay#2/Assay#1, whichallows for determination of Ca-2 cancer isoform levels.

To evaluate the levels of BIN1 cancer isoform Ca-2, BIN1 Cancer Test #1was used (ratio Assay#2/Assay#1). FIG. 6 shows the BIN1 Cancer Test #1results, related to subject age in normal blood samples. Three normalsamples showed results above 10 (i.e. at the Hatch marks on the y-axis).These samples derive from younger patients between 25 and 43 years old.Nearly all the normal samples have a very low Cancer Test #1 values.

In FIG. 7, the BIN1 Cancer test #1 was used to evaluate the cancersamples and these results are plotted with the results obtained with thenormal samples. A test value above of 10 was observed in ten cancersamples including lung, pancreas, ovarian and thyroid cancer. Lungcancer showed the highest levels of Ca-2 (exon 12a+/13−BIN1), bothquantitatively and numerically, thus five of ten samples were positiveand these were among the highest levels observed. The levels of Ca-2BIN1 cancer isoform in colorectal cancer were uniformly very low, whilea few samples from subjects with each of the other cancer types did showlevels above 10; including two pancreas samples, two ovarian samples andone thyroid samples.

The youngest cancer patient with a Test value above 10 in this smallsurvey was 56 years old, whereas among the three normal subjects withelevated BIN1 test values, the oldest is 43 years old. Thus in the agegroups in which one is most likely to consider a cancer screening test,the separation of the test results in the cancer samples and normals isvery good at a level of 10: 20% of cancers are positive, and no normalsare positive in persons >50 years old. Half of the lung cancer sampleswere positive suggesting the use of a BIN1 test to screen smokers, andothers with a high risk of lung cancer. Interestingly, three of theselung cancers that were detected were Stage I, thus these patients maygain significant benefit from early detection and directed treatment.

Table 4 summarizes the human clinical results using BIN1 Cancer Test #1and an increasing threshold of test signal. (* these normals are <=43years old, ̂ these cancer subjects are >=56 years old).

TABLE 4 Percentage of detection of BIN1 cancer isoform in Normal andCancer samples. BIN1 Cancer Test #1 % of Normal % of Cancer   >1 9.528.0  >10 7.1* 20.0{circumflex over ( )}  >100 4.8 14.0 >1000 2.410.0 >1.00E+04 2.4 6.0 >1.00E+05 2.4 6.0 >1.00E+06 2.4 6.0 >1.00E+07 2.44.0 >1.00E+08 2.4 2.0 >1.00E+09 0.0 2.0 *these normals are ≦43 years old{circumflex over ( )}these cancer subjects are ≧56 years old

FIG. 8 represents the BIN1 Cancer Test #1 results in cancer patientsrelated to the different stages of cancer progression (I to IV) for allthe cancer samples used in this study. An overall increase in BIN1cancer test signal was observed in stage IV compared to stage III. FIGS.9A-9E represent the test results related to the cancer stages, detectedin each type of cancers. In lung cancer, two samples had high BIN1cancer test results, one stage I and stage IV (FIG. 9A). In pancreascancer, two stage IV cancer patients had relatively high Bin 1 cancertest results compared to the stage III cancer patients (FIG. 9B). Inovarian cancer, two samples had high Bin 1 cancer test results; onestage I and stage IV (FIG. 9C). Finally in thyroid cancer, the Bin 1cancer test results were highest in a stage II cancer sample (FIG. 9E).

Thus, the BIN1 Cancer test detects a subset of cancer samples, inseveral cancers. These subsets may correspond to stage but do notnecessarily correlate with stage. The BIN1 positive subsets may reflecttumor subtypes within these cancer diagnoses, i.e., subtypes that mayhave different biochemical pathways active or different host responsesoperating. This suggests that BIN1, by identifying subsets in lung,ovarian, thyroid, and pancreatic cancer, may be useful for treatmentselection in these patients.

Example 4 BIN1 Detection in Blood Samples from Dogs Treated for CancerMaterials and Methods

Dog samples.

Fourteen blood samples from apparently healthy dogs (referred to asNormal) and forty three blood samples from dogs newly diagnosed withcancer (referred to as Cancer/Pre) were collected for this study.Several dogs underwent treatment, including surgical resection, and/orradiotherapy, and chemotherapy. A second blood sample was obtained froma subset of treated dogs one week and in some cases two weeks aftertreatment (Post1 and Post2, respectively).

Results

BIN1 Cancer Test #1 was used to analyze 57 dog samples (14 normals and43 with new diagnosis) detect the levels of BIN1 cancer isoform innormal versus cancer dog samples, pre- and post-treatment. No detectionof BIN1 cancer isoform was detected in normal samples (FIG. 10).Elevated BIN1 cancer isoform was detected in cancer samples. The levelsof BIN1 cancer isoform decreased after cancer treatment (FIG. 10).

Fourteen pretreatment cancer samples showed elevated BIN1 cancer testresults, with levels greater than 1, compared to normal (all normalswere zero). Following cancer treatment, the level of BIN1 cancer isoformwas decreased in eight out of eleven samples (FIGS. 11A-11C). No changein the level of BIN1 cancer isoform was observed in dog sample #4, #7,and #8 after treatment. Dog #4 had oral sarcoma with apparently clean 2cm margins on surgical resection. However the high post-surgical BIN1levels were suggestive of persistent disease, and the follow-uphistopathology indicated that the margins were not clean and thecancerous cells had a high mitotic index. Treatment priority was shiftedto palliative care and no further surgery was performed. Dog #7presented with thyroid carcinoma that was surgically removed, butsurgical and histopathology analysis showed vascular invasivemetastasis, suggesting inadequate treatment. Dog #8 presented with amast cell tumor that had poor response to therapy and the dog was latereuthanized as the cancer continued to spread. Therefore, the high levelsof BIN1 cancer isoform detected in blood samples correlated with cancerprogression or with the absence of response to cancer treatment.

1. A method of screening for cancer in a subject comprising: (a)obtaining a blood sample from the subject; (b) detecting a level of12a+Bridging Integrator 1 (BIN1) polypeptide in the sample; (c)detecting a level of 12a+/13+ BIN1 polypeptide in the sample; (d)comparing the detected level of step (b) to the detected level of step(c) to determine a 12a+/13− BIN1 value for the sample; (e) comparing the12a+/13− BIN1 value from step (d) to one or more control values, whereinan elevated 12a+/13− BIN1 value indicates the subject has a cancer; and(f) obtaining a tissue sample from the subject identified in step (e).2. The method of claim 1, wherein an elevated 12a+/13− BIN1 value isbased on a threshold value determined by comparing a negative controlvalue to one or more positive control values, wherein an 12a+/13− BIN1value greater than the threshold value indicates the subject has acancer.
 3. The method of claim 2, wherein the threshold value is set atleast one standard deviation above a mean negative control value. 4.(canceled)
 5. The method of claim 2, wherein the threshold value showsno statistically significant difference as compared to a positivecontrol value. 6-9. (canceled)
 10. The method of claim 1, wherein thesubject is at risk of developing cancer.
 11. The method of claim 10,wherein the subject is at risk of developing lung cancer.
 12. (canceled)13. The method of claim 1, wherein the 12a+/13− BIN1 value and the oneor more control values are normalized for the age of the subject. 14.The method of claim of claim 1, wherein the subject is at least 50 yearsof age.
 15. The method of claim 1, wherein 12a+ BIN1 polypeptides aredetected using an 12a+ BIN1 specific antibody.
 16. The method of claim15, wherein the antibody selectively binds SEQ ID NO:1 but does not bindSEQ ID NO:3.
 17. The method of claim 16, wherein the antibody is amonoclonal antibody or a recombinant antibody.
 18. The method of claim17, wherein the complementarity determining regions (CDRs) of theantibody heavy chain comprise the amino acid sequences SEQ ID NO:10, SEQID NO:11, and SEQ ID NO:12.
 19. The method of claim 17, wherein the CDRsof the antibody light chain comprise the amino acid sequences SEQ IDNO:13, SEQ ID NO:14, and SEQ ID NO:15.
 20. A method of determiningefficacy of a cancer therapy in a subject, the method comprising: (a)obtaining a first blood sample from a subject with cancer prior totreatment with a first cancer therapy; (b) determining a first 12a+/13−BIN1 value in the first blood sample; (c) obtaining a second bloodsample from a subject with cancer after at least one treatment with thefirst cancer therapy; (d) determining a second 12a+/13− BIN1 value inthe second blood sample; (e) comparing the first 12a+/13− BIN1 value tothe second 12a+/13− BIN1 value; and (e) selecting an second cancertherapy for the subject if the 12a+/13− BIN1 value increases or fails todecrease in the second blood sample as compared to the first bloodsample or continuing to treat the subject with the first cancer therapyif the 12a+/13− BIN1 value decreases in the second blood sample ascompared to the first blood sample. 21-24. (canceled)
 25. A method oftreating cancer in a subject comprising (a) obtaining a blood samplefrom the subject; (b) detecting a level of 12a+ Bridging Integrator 1(BIN1) polypeptide in the sample; (c) detecting a level of 12a+/13+ BIN1polypeptide in the sample; (d) comparing the detected level of step (b)to the detected level of step (c) to determine an 12a+/13− BIN1 valuefor the sample; (e) comparing the 12a+/13− BIN1 value from step (d) toone or more control values; and (f) administering to the subject aninhibitor of indoleamine 2,3-dioxygenase (IDO) if an elevated 12a+/13−BIN1 value is determined.
 26. The method of claim 25, wherein anelevated 12a+/13− BIN1 value is based on a threshold value determined bycomparing a negative control value to one or more positive controlvalues, wherein an 12a+/13− BIN1 value greater than the threshold valueindicates the subject has a cancer.
 27. The method of claim 26, whereinthe threshold value is set at least one standard deviation above a meannegative control value.
 28. The method of claim 26, wherein thethreshold value is set at least two standard deviations above a meannegative control value.
 29. The method of claim 26, wherein thethreshold value shows no statistically significant difference ascompared to a positive control value. 30-33. (canceled)
 34. The methodof claim 25, wherein the level of exon 12a+ BIN1 polypeptide isdetermined using a 12a+ BIN1 specific antibody.
 35. The method of claim29, wherein the antibody selectively binds SEQ ID NO:1 but does not bindSEQ ID NO:3.
 36. The method of claim 34, wherein the antibody is amonoclonal antibody or a recombinant antibody.
 37. The method of claim36, wherein the complementarity determining regions (CDRs) of theantibody heavy chain comprise the amino acid sequences SEQ ID NO:10, SEQID NO:11, and SEQ ID NO:12.
 38. The method of claim 36, wherein the CDRsof the antibody light chain comprise the amino acid sequences SEQ IDNO:13, SEQ ID NO:14, and SEQ ID NO:15.
 39. The method of claim 25wherein the IDO inhibitor comprises 1-methyl-tryptophan or1-methyl-D-tryptophan.
 40. An isolated antibody that selectively bindshuman 12a+ BIN1, wherein the antibody binds SEQ ID NO:1 but does notbind SEQ ID NO:3.
 41. The isolated antibody of claim 40, wherein thecomplementarity determining regions (CDRs) of the antibody heavy chaincomprise the amino acid sequences SEQ ID NO:10, SEQ ID NO:11, and SEQ IDNO:12.
 42. The isolated antibody of claim 40, wherein the CDRs of theantibody light chain comprise the amino acid sequences SEQ ID NO:13, SEQID NO:14, and SEQ ID NO:15. 43-44. (canceled)
 45. A kit for determining12a+/13− BIN1 isoform levels comprising: (a) an assay system fordetecting 12a+ BIN1 polypeptide comprising (i) an antibody thatselectively binds 12a+ BIN1, and (ii) an antibody that selectively bindsmultiple human BIN1 isoforms, wherein, if the antibody of (a)(i) isimmobilized on a solid surface, the antibody of (a)(ii) is notimmobilized, or, if the antibody of (a)(i) is not immobilized, theantibody of (a)(ii) is immobilized on a solid surface; and (b) an assaysystem for detecting 12a+/13+ BIN1 polypeptide comprising (i) anantibody that selectively binds 13+ BIN1, and (ii) an antibody thatselectively binds 12a+ BIN1, wherein, if the antibody of (b)(i) isimmobilized on a solid surface, the antibody of (b)(ii) is notimmobilized, or, if the antibody of (b)(i) is not immobilized, theantibody of (b)(ii) is immobilized on a solid surface.
 46. A kit ofclaim 45, wherein the antibody of (a)(ii) selectively binds 11+ BIN 1.